Ophthalmic composition containing n-acetylcysteine for the treatment of dry-eye syndrome

ABSTRACT

An ophthalmic pharmaceutical composition containing acetylcysteine as the active ingredient having a mucolitic action, suitable for administration in eyedrops, where N-acetyl-cysteine is neutralised with base DEAE-dextrane in order to reach pH levels preferably between 6.0 and 7.5, and having a physiologically acceptable osmolarity less than 320 mOsm/kg. The presence of a neutralising agent for N-acetyl-cysteine that does not negatively affect the composition osmolarity allows the formulation to be isotonic or even hypotonic, thus avoiding the irritative and potentially damaging effects of previous ophthalmic products containing N-acetyl-cysteine.

[0001] The present invention relates to an ophthalmic compositioncontaining N-acetyl-cysteine for the treatment of dry-eye syndrome. Moreparticularly, the invention concerns compositions containingN-acetyl-cysteine as an active ingredient, which are suitable fortopical administration in eyedrops because they are isotonic with thelacrimal fluid thanks to the presence of a neutralising agent forN-acetyl-cysteine that has no adverse effects on the osmolarity of thecomposition.

[0002] As is known, preocular tear film is an organised liquid structurecovering the conjunctiva and the exposed surface of the eyeball. Undernormal conditions, the tear film appears as a complex three-layeredstructure, consisting of:

[0003] an internal mucous layer, composed of a glycoprotein mixture(mucin) produced by specialised cells (goblet cells) present in theconjunctival epithelium—this layer adheres to the corneo-conjunctivalepithelium forming a hydrophilic surface.

[0004] an abundant intermediate aqueous layer, overlaying the aforesaidhydrophilic surface, rich in oxygen and essentially composed of water,electrolytes, proteins, enzymes and mucin, that has a trophic, defence,vehicular and optical function.

[0005] a thin external lipid layer that mainly acts to regulate theevaporation rate of water from the tear film.

[0006] The aforesaid three-layered structure constitutes a complexphysiological structure whose main functions are those of protecting theeye surface, maintaining the hydration, lubrication and cleanliness ofthe corneal surface, and cooperating in producing proper eye-sight. Theperfect equilibrium and continuous renewal of this physiological systemis a necessary condition for it to fulfil its functions. For this tooccur, there must firstly be a constant but not excessive waterevaporation from lacrimal fluid, such that it maintains osmolarityaround a physiological value of 300 mOsm/kg, and the tear film must becontinuoulsy redistributed on the corneal surface through blinking.

[0007] Various pathological or anomalous situations of the eye appearthrough discontinuity or alterations of the tear film, as a result, forexample, of an inadequate blinking rate, prolonged use of contactlenses, the use of certain systemic pharmaceutical drugs or of senilehyposecretion. In this context, the term “dry-eye syndrome” is taken tomean a set of pathological manifestations of the ocular areacharacterised by the reduction and/or altered composition of the tearfilm, while more appropriately the characteristic alterations of thecorneal surface that are produced in this way are identified with theterm keratoconjunctivitis sicca. As may be self-evident, dry eye is aclinical disorder that is difficult to define as regards frequencybecause it is not always recognised due to its often bland and aspecificsymptomatology and objectivity (Lemp M. A., Recent developments in dryeye management, Ophthalmology 10, 1299-304 (1987); Lemp M. A., Dry eyesyndromes: treatment and clinical trials. Adv. Exp. Med. Biol., 350553-9 (1994); Lemp M. A., Epidemiology and classification of dry eye,Adv. Exp. Med. Biol., 438, 791-803 (1998)). A patient with dry-eyesyndrome presents disorders such as conjunctival reddening, difficultyin opening eye-lids on awakening, a burning, prickling and sandysensation, a feeling of a foreign body and also photophobia. Possiblecomplications range from keratopathy with surface disepithelisation toinfective keratitis and even serious infective degenerative pathologiesof the cornea.

[0008] From a diagnostic standpoint, besides an evaluation of its ownkind of symptoms, dry-eye syndrome may be identified and monitoredthrough consolidated procedures, the most common being the measurementof tear production (the Schirmer test), tear film break-up time (BUT)after blinking and an evaluation of eye surface staining using rosebengal or fluorescein. Moreover, the value of tear osmolarity may betaken as an objective parameter of the pathological state underexamination, it being demonstrated that in pathological conditions thisvalue increases by 30-40 mOsm/kg on average.

[0009] The possible etiological factors are many. It is sometimes notpossible, even with laboratory investigations and sophisticatedequipment, to recognise the cause of this syndrome; in this case, onetalks in terms of an “essential form”, which is treatable exclusively asa symptom. In view of the fact that tear film alterations may havedifferent pathological interpretations and therapeutical approachesdepending on the type of the lacrimal component that is found altered, afundamental step consists of assessing whether the anomaly is located inthe lipid surface component, in the aqueous layer or in the internalmucinic one (Miglior M., Troiano P., Lacrimal film pathologies:classification and rationale of the therapy, in Symposium on theLacrimal System, Toronto, Jun. 25, 1994, ed. Hurwitz J. et al., Kugler &Ghedini Publ., (1995)), to then draw the more appropriate conclusionsfrom a therapeutic standpoint.

[0010] The alteration of the external lipid component, that is not perse a very frequent case, is usually due to a blepharitis, and must betreated as such. In the more frequent case that the intermediate aqueouslayer is altered, this alteration always consists of a quantitativereduction of this component, linked to excessive evaporation, that leadsto the aforesaid increase in lacrimal osmolarity. In this caseparticular compositions in eyedrops are indicated, known as “artificialtears”, in formulations rich in water and with few or no mucomimeticagents, possibly associated with low hydration corneal contact lenses.

[0011] When the alteration is in the internal mucin layer, then the caseis particularly delicate. It is known that the integrity of the mucinlayer is one of the essential factors in maintaining tear film stabilitybecause the mucin improves the wettability of the corneal surface,allows the aqueous film to continuously and homogeneously adhere to theexposed surface, assuring stability, and increases the fluid phaseviscosity, preventing too rapid a flow from the conjunctival sac.

[0012] As regards the mucin layer alterations, it has been reported(Miglior M. et al., loc. cit.) that they may appear with either a mucusdeficit or an excess. When mucin is absent or insufficient, the corneabecomes unwettable and, due to the imbalance between the electrolytesand glycoproteins present, the tear film becomes unstable and can breakup, thus producing dry areas. In the second case, an excess of mucincoagulates into threads that still hinder ocular surface wettability andleads to the formation of dry areas and to damage to the cornealepithelium. In this situation, too, it is possible to intervene at atopical ocular level by using suitably formulated “artificial tears”additioned with appropriate integrators of the mucin component andmucomimetic agents (namely, various cellulose derivatives, polyvinylderivatives such as polyvinylpyrrolidone and polyvinyl alcohol, variouspolysaccharides and their derivatives, such as dextrane, hyaluronicacid, chondroitin sulfate, etc.) or, in case of an excess of the mucincomponent, using mucolitic agents such as N-acetyl-cysteine.

[0013] More specifically, for lacrimal mucous reduction andfluidification, Nacetyl-cysteine is mainly prescribed both for topicaland systemic administration. Alternatively, other mucolytics can beused, such as carboxymethyl cysteine, bromhexine and ambroxol (thelatter two with also a not better defined secretion-stimulating action)for systemic administration. The first of the aforesaid activeingredients, having the following structural formula,

[0014] is a derivative of the natural amino acid L-cysteine that provesto be clinically useful as a mucolitic agent in acute and chronicbronchopulmonary pathologies, and is thus mainly prescribed for thesedisorders, mostly using a systemic administration. This molecule isconsidered to exert its activity by “breaking up” the disulfide (S—S)bonds of the mucus, thus reducing its viscosity. In the eye,N-acetyl-cysteine solutions can dissolve the mucus threads therebyreducing lacrimal viscosity.

[0015] N-acetyl-cysteine (also referred to hereinafter as N-AC) is arelatively strong acid and cannot be directly applied to the ocularsurface as such, but only after being suitably neutralised. For example,a 4% weight N-AC aqueous solution has a pH of 1.90, and before it can beused in eyedrops, the acid form must be neutralised, for example withNaOH, in order to yield the corresponding sodium salt and to bring thepH within a range that is physiologically tolerated by the human eye.However, the necessary neutralisation has the drawback of increasing theosmolarity of the resulting solution that goes from an original value of241 mOsm/kg to values corresponding to high hypertonicity. In thisregard, Italian patent No. 1151755 (Bruschettini s.r.l.) describes aneyedrop composition containing N-AC, where the active ingredient issupplemented with a disodium phosphate buffer and an amount of sodiumbicarbonate sufficient to bring the pH of the solution within the range6.6-7.0. Even though the document does not highlight any resultinginconveniences in tolerability, it presents hypertonicity as afundamental characteristic of the proposed formulation.

[0016] Another N-acetyl-cysteine based formulation for topicalophthalmic use is described in EP-A-0 551 848 (Zambon Group S.p.A.), inwhich the N-AC solution, preferably 4% weight, is associated withpolyvinyl alcohol in order to obtain a product of improved activity forthe treatment of keratoconjunctivitis sicca. In this case, the documentdoes not report the osmolarity values presented by the proposedformulations, but it is clear from the corresponding compositions thatthey are strongly hypertonic formulations.

[0017] Actually, currently commercially available N-AC-based eyedropspresent much higher osmolarity values than the “physiological” value of300 mOsm/kg. These values are >1000 mOsm/kg for the product marketedunder the Brunac® tradename (Bruschettini s.r.l., containing 5% N-AC),and around 900 mOsm/kg for the Tirocular® product (ACRAF S.p.A,containing 4% N-AC). As previously pointed out as regards the importanceof avoiding excessive tear film hypertonicity, it is obvious that theaforesaid formulations may be irritating and potentially damaging to thecorneal surface. This is all the more so if it is considered thatlacrimal fluid of patients with dry-eye syndrome is already hypertonicand thus the relative treatment must not aggravate these conditions byadministering hypertonic preparations. It is for these veryconsiderations that artificial tears are produced in isotonic or,preferably, hypotonic formulations (Holly F. J., Lamberts D. W., Effectof nonisotonic solutions on tear film osmolality. Invest. Ophthalmol.Vis. Sci., 20, 236 (1981)).

[0018] Considering also the possibility of neutralising acetylcysteinewith organic bases other than an inorganic NaOH base, stronglyhypertonic solutions are still obtained, as the following examples show.Neutralisation with sodium hydrate N-AC 5.00 g Viscosity enhancingpolymer 1.00 g NaOH 1.18 g Sterile dist. H₂O q.s. to 100 ml pH = 7.00;Osmolarity = 644 mOsm/kg

[0019] Neutralisation with L-lisine N-AC 5.00 g L-lisine 4.50 g Sodiumedetate 0.10 g Viscosity enhancing polymer 2.00 g Sterile dist. H₂O q.s.to 100 ml pH = 7.00; Osmolarity = 620 mOsm/kg

[0020] Neutralisation with L-arginine N-AC 5.00 g L-arginine 6.20 gSodium edetate 0.10 g Viscosity agent polymer 2.00 g Sterile dist. H₂Oq.s. to 100 ml pH = 7.00; Osmolarity = 656 mOsm/kg

[0021] Neutralisation with L-istidine N-AC 4.00 g L-istidine 8.00Sterile dist. H₂O q.s. to 100 ml pH = 6.50; Osmolarity = 409 mOsm/kg

[0022] Neutralisation with L-methylglucamine N-AC 4.00 gL-methyl-glucamine 3.40 g Sterile dist. H₂O q.s. to 100 ml pH = 7.30;Osmolarity = 490 mOsm/kg

[0023] Neutralisation with L-glycine sodium carbonate N-AC 4.00 gL-glycine sodium carbonate 3.60 g Sterile dist. H₂O q.s. to 100 ml pH =7.20; Osmolarity = 1636 mOsm/kg

[0024] Neutralisation with L-glucamine N-AC 4.00 g L-glucamine 4.80 gSterile dist. H₂O q.s. to 100 ml pH = 7.30; Osmolarity = 631 mOsm/kg

[0025] The foregoing examples show that commonly used pharmaceuticalneutralisers lead to considerably hypertonic formulations in cases of5.0% or 4.0% N-acetyl-cysteine, and both in the presence or absence of aviscosity enhancing polymer.

[0026] In view of the above, it is an object of the present invention toprovide an acetylcysteine-based pharmaceutical composition for topicalophthalmic use which can be advantageously used in mucolitic activepreparations for the treatment of dry-eye syndrome without presentingthe drawbacks of poor tolerability or toxicity for ocular tissuesnormally linked to low pH values and, above all, to osmolarity valuesmuch higher than physiological ones. More specifically, the ophthalmicpreparation—although exploiting the mucolitic properties ofacetylcysteine—must be chemically close to neutrality and, at the sametime, be isotonic or, preferably, hypotonic.

[0027] To this end, the present invention proposes neutralising theacetylcysteine with a base agent that, although positively contributingto the overall performance of the formulation as an artificial tear,also poorly contributes to tonicity, thus allowing to obtain N-ACsolutions which are isotonic, or even hypotonic, with lacrimal fluid.

[0028] Within the frame of the studies that have led to the presentinvention, it has been found that a polycationic derivative of dextrane,diethylaminoethyldextrane (DEAE-dextrane), containing diethylaminoethylgroups linked to the glucose moieties of the dextrane skeleton throughether bonds that are exceptionally stable to acid hydrolysis, issurprisingly effective in neutralising the acidity of N-acetyl-cysteineby forming its corresponding salt, without appreciably contributing tothe tonicity of the resulting solution. At the same time, theformulation obtained by neutralising N-AC with the polycationicderivative of dextrane already incorporates a viscosity enhancingpolymer whose presence, as already noted, is advantageous in a greatmany disorders linked to dry-eye syndrome.

[0029] DEAE-dextrane, which is already known and used in medicine forthe treatment of hypercholesterolemy and as an antilipermic in general(U.S. Pat. Nos. 3,627,872 and 4,160,826), is obtained by reacting2-chloroethyldiethylamine, in an alkaline solution, with dextrane—apolysaccharide whose structure formula may be illustrated as follows:

[0030] As the starting polysaccharide, the diethylaminoethyl derivativeis composed of glucopyranose units linked mainly through 1→6 bonds,while a small number of 1→3 bonds (and, to a lesser extent, 1→2 and 1→4bonds) are responsible for the side branching. The average molecularweight is about 500,000 and the nitrogen content is approximately 3.2%,which corresponds to one cation group for every three glucose units.Similarly to dextrane sulphate, DEAE-dextrane is a polyelectrolyte andits properties differ considerably from the starting polymer—dextrane.

[0031] Being a polycationic product, DEAE-dextrane is normallycommercially available in the salified form of the correspondingchloride or sulphate. The free base form, necessary as a startingreagent for neutralising N-acetylcysteine as proposed according to thepresent invention, may be prepared, for example, from DEAE-dextranechloride (or sulfate) by treating with ionic exchange resins, accordingto the following method.

[0032] A ionic exchange resin (DOWEX 1×8, 30 ml) is packed in a columnand treated with 0.1N NaOH in order to hydrate and activate it. Next, 1NNaOH is made to pass through the resin. The resin is then washed atlength in water to eliminate the excess NaOH and a 5% DEAE-dextrane HCLsolution is introduced in the column. The solution in the column,containing base DEAE-dextrane, is concentrated by reduced pressureevaporation and then dried.

[0033] To determine the neutralising capacity of N-acetyl-cysteine withwhich it must be formulated, the product in a free base form asobtained, if required, from the previous operation is titrated with 0.1NHCl. This operation, which allows a preventive determination of thenumber of chemical equivalents contained in the mass unit of theneutralising agent, may be carried out, for example, by a “returntitration” in which to a certain quantity of base DEAEdextrane dissolvedin distilled water is first added an excess of strong acid (HCl) andthen the excess strong acid is titrated with a strong base (NaOH). ThepH values, measured via potentiometrically), obtained with thisoperation, are reported in one of the attached diagrams. By way ofexample and with reference to certain specific embodiments of theinvention,

[0034]FIG. 1 shows a titration curve of the base DEAE-dextrane used inthe composition of the present invention;

[0035]FIG. 2 shows the results of the Schirmer test in an evaluation ofthe activity of the composition in an experimental model of dry-eyesyndrome in rabbit; and

[0036]FIG. 3 shows the results of the observation via slit lamp of thecornea after staining with sodium fluorescein in the same experimentalmodel in rabbit.

[0037] Temporarily leaving aside the two figures concerning theapplication experimentation of the compositions of the invention, thatwill be dealt with later on, and returning to the base DEAE-dextranetitration, FIG. 1 shows the potentiometric curve obtained when using theaforesaid procedure to treat 0.08 g of base DEAE-dextrane dissolved in 5ml of distilled water, to which was added 5.0 ml of N/10 HCl. Thepotentiometric titration of the excess HCl with NaOH, in which the pHvalues progressively obtained are indicated in the diagram, showed thatthe flexion point at pH 7.0 is reached after adding 3.6 ml of basicsolution. Therefore, 1.4 ml (5.0-3.6) of N/10 HCl, i.e. 0.14 mEq,neutralised 0.08 g of base DEAE-dextrane. This allows assessing theequivalent weight of the polycationic reagent under study as0.14/0.08=1.75 mEq/g.

[0038] In view of the above, it is thus possible to easily realise aN-AC-based formulation in which this active ingredient is neutralised byan appropriate proportion of base DEAE-dextrane that, as will be seenmore clearly in the following examples, does not penalise the osmolarityof the composition but allows obtaining ophthalmic solutions that arealso advantageously isotonic.

[0039] Therefore, the present invention specifically provides anophthalmic composition based on acetylcysteine for administration ineyedrops, containing N-acetyl-cysteine neutralised with baseDEAE-dextrane and having a physiologically acceptable osmolarity. Morespecifically, as is already known, said osmolarity is less than 320mOsm/kg, and preferably less than 300 mOsm/kg, while the pH of thecomposition ranges between 6.0 and 7.5, and is preferably in the range6.2-7.0. The above characteristics allow achieving solutions which maybe administered in eyedrops suitable for the treatment of dry-eyesyndrome—the solutions preferably containing from 3 to 5% weightN-acetyl-cysteine neutralised with a quantity of between 9% and 15% byweight of base DEAE-dextrane.

[0040] The formulations according to the present invention areconveniently prepared as either a solution or an aqueous suspension in apharmaceutically acceptable ophthalmic vehicle, and may contain one ormore of the other possible ingredients known in pharmaceuticaltechnology for this kind of preparations. In particular, in addition toN-acetyl-cysteine and DEAEdextrane, the formulations may also containminor quantities of other viscosity enhancing polymers, such as the onescited with reference to the prior art and of which some are reported inthe example formulations below. As is customary, the compositions maycontain further adjuvants, among which chelating agents, antimicrobialsand preservatives, although the latter are normally avoided in eyedrop,if possible by using unitary dose packages without preservatives.

[0041] Some specific embodiments of the ophthalmic compositionsaccording to the present invention, as well as some experimental dataconcerning the performance of these compositions and a comparison withthe prior art, are reported in the following examples.

EXAMPLE 1

[0042] Taking into account the equivalent proportions already determinedas previously described, a N-acetyl-cysteine and DEAE-dextrane basedhypotonic preparation is produced with the following formulation: N-AC4.0 g Base DEAE-dextrane 12.0 g Sterile dist. H₂O q.s. to 100 ml pH =6.18; Osmolarity = 245 mOsm/Kg

[0043] For the preparation, 4 g of NAC is dissolved in about half thequantity of water available and 12 g of base DEAE-dextrane is dissolvedin the remaining water. The two solutions are then combined and filteredusing a 0.2 pm sterilising filter.

[0044] In addition to the aforesaid osmolarity value, the compositionshows a viscosity of 21 mpa-s and a Newtonian type flow.

EXAMPLE 2

[0045] The hypotonic composition according to the invention in this casecontains a preservative, according to the following formulation: N-AC4.0 g Base DEAE-dextrane 12.0 g Benzalkonium chloride 0.01 g Steriledist. H₂O q.s. to 100 ml pH = 6.23; Osmolarity = 245 mOsm/kg

[0046] For the preparation, N-AC, DEAE-dextrane and benzalkoniumchloride are separately dissolved in distilled water. The solutions arethen combined and filtered using a 0.2 μm sterilising filter.

[0047] If required, the osmolarity may be brought to a physiologicalvalue of 300 mOsm/kg by adding NaCl.

EXAMPLE 3

[0048] Taking into account the already determined equivalent proportionsas described above, and proceeding for the preparation in a similarmanner as the examples illustrated above, a N-acetyl-cysteine andDEAE-dextrane based hypotonic preparation is produced according to thefollowing formulation: N-AC 3.0 g Base DEAE-dextrane 9.0 g Sterile dist.H₂O q.s. to 100 ml pH = 6.2; Osmolarity = 185 mOsm/Kg

EXAMPLE 4

[0049] A hypotonic but more concentrated preparation is obtained with asimilar preparatory procedure, but with the following quantities: N-AC 5.0 g Base DEAE-dextrane 15.0 g Sterile dist. H₂O q.s. to 100 ml pH =6.5; Osmolarity = 307 mOsm/Kg

[0050] Other formulation examples that show the optional presence offurther viscosity enhancers, whose concentration generally rangesbetween 0.5 and 3% weight, are reported below. In all the cases, theprocedure for the preparation is similar to the ones illustrated in theprevious examples, and hypotonic products are obtained in all cases.

EXAMPLE 5

[0051] N-AC 4.0 g Base DEAE-dextrane 12.0 Polyvinyl alcohol*  0.5Sterile dist. H₂O q.s. to 100 ml

EXAMPLE 6

[0052] N-AC  4.0 Base DEAE-dextrane 12.0 Hydroxypropyl cellulose*  0.4Sterile dist. H₂O q.s. to 100 ml

EXAMPLE 7

[0053] N-AC 4.0 g Base DEAE-dextrane 12.0 Hyaluronic acid*  0.2 Steriledist. H₂O q.s. to 100 ml

EXAMPLE 8

[0054] N-AC 4.0 g Base DEAE-dextrane 12.0 Polyvinylpyrrolidone*  0.4Sterile dist. H₂O q.s. to 100 ml

[0055] In order to assess the performance of the compositions accordingto the present invention, several experiments were carried out and someof the results are reported below.

[0056] Biocompatibility Studies

[0057] Biocompatibility studies were carried out in vivo with rabbiteyes by administering 50 μl of the formulation described in Example 2,at close intervals. No primary signs of irritation were found.

[0058] Stability Studies

[0059] The formulations underwent autoclaving (120° C., 20 min.): ashighlighted in the prior art, N-acetyl-cysteine is degraded by about 30%(both in the presence and absence of benzalkonium chloride). It is,however, possible to carry out sterilisation by filtering through a 0.2pm membrane, as was performed in the aforesaid examples.

[0060] Analytical Method used for Determining N-AC in the Presence ofDEAE-dextrane

[0061] In order to evaluate the stability of N-AC in the vehicle, it isnot possible to use the HPLC method or ultraviolet spectrophotometry.The following calorimetric method was thus employed (Raggi M. A.,Cavrini V. and Di Pietra A. M., Colorimetric determination ofacetylcysteine, penicillamine and mercaptopropionylglycine inpharmaceutical dosage forms, J. Pharm. Sci., 71, 1384 1386 (1982)).

[0062] Reagents Used

[0063] 1,10-phenantroline 0.25% (100 ml)

[0064] Ferric solution 4×10⁻³M (1000 ml)

[0065] pH 4 acetate buffer (100 ml)

[0066] Sodium acetate 0.2 M (100 ml)

[0067] Reagent Preparation

[0068] o-phenantroline 0,25%

[0069] 0.25 g of the compound is dissolved in distilled H₂O and afterslight heating is then taken to the required volume with distilled H₂O.The solution is stored for three days in a dark glass container awayfrom the light.

[0070] Ferric Solution

[0071] 1.92 g of FeNH₄SO₄ ×12H ₂O is dissolved in distilled H₂O and thentreated with 10 ml of concentrated HCl. It is taken to the requiredvolume with distilled H₂O. The solution is stored for three days in adark glass container away from the light.

[0072] pH 4 Buffer

[0073] This is obtained by mixing 75 ml of 0.1M CH₃COOH with 25 ml of0.1M CH₃COONa. If necessary, the pH may be adjusted with a solution of0.2M CH₃COONa.

[0074] Standards Preparation

[0075] 0.163 g of acetylcysteine is dissolved in 500 ml of distilledH₂O. By diluting in a proportion of 1:5, the required concentration isobtained.

[0076] Increasing volumes of this solution (1-5 ml) are placed in a 25ml flask and then the following are added in succession:

[0077] 6 ml of ferric solution

[0078] 2.5 ml of o-phenantroline 0.25%

[0079] 3.5 ml of 0.2M NaOAc

[0080] 4.5 ml of pH 4 buffer

[0081] After 20 min the adsorbance is measured at 515 nm by using asreference a control prepared at the same time as the sample.

[0082] The procedure described above was also applied to carry out thecalibration of N-AC in the presence of DEAE-dextrane. For these samples,the measurement was carried out by using as reference a controlcontaining the polymer and also a control without DEAE-dextrane. Thespectra are comparable.

[0083] By comparing the spectrum obtained with N-AC with the oneobtained for the samples also containing the polymer, it may beconfirmed that DEAEdextrane does not interfere with the adsorption, butit modifies its intensity.

[0084] Activity Studies in an Experimental Model of Dry-Eye Syndrome inthe Rabbit.

[0085] The trials were carried out on a group of 10 New Zealand albinomale rabbits weighing 2-2.5 kg and kept in standard conditions. Theformulation of Example 2 (called DEAE/N-AC) was compared not only withcontrols that only received a physiological solution, but also with acommercially available (hypertonic) formulation containing 4% N-AC.

[0086] A drop of a solution of 1.0% atropine sulphate (AS) wasadministered in both eyes of the animals 3 times a day for 5 consecutivedays in order to cause an experimental dry-eye condition (Burgalassi S.,Panichi L., Chetoni P., Saettone M. F. and Boldrini E., Development of asimple dry eye model in the albino rabbit and evaluation of some tearsubstitutes, Ophthalmic Res. 31, 229-235 (1999)). 5 minutes after ASadministration, 50 μl (corresponding to one drop) of one of theformulations under study or of a physiological solution (control group)was instilled in only the right eye.

[0087] At appropriate time intervals (2, 3, 4 and 5 days from the startof the treatment) the animals underwent the Schirmer test and anobservation of the ocular surface, after staining with sodiumfluorescein, by slit lamp with a cobalt blue filter.

[0088] The Schirmer test envisages the introduction of a strip ofblotting paper of standard size and materials in the external third ofthe lower conjunctival formix. The strip is left in place for a fixedperiod of time (3 min). The time taken for the lacrimal fluid to riseand the length in mm of the portion of paper wetted by the tears providethe score for lacrimal secretion. The test results obtained in thevarious treatment conditions are reported in FIG. 2. The vertical axisof the graph gives the millimetres of wetted strip in three minutes. Theunbroken line (baseline) corresponds to the average physiological value,observed in the untreated animals (21,2 mm).

[0089] It may be noted that, in the eyes treated with a physiologicalsolution (control group), there is a clear decrease in lacrimalsecretions, that continues over the five days of observation. TheSchirmer test scores for the hypertonic commercially availableformulation do not greatly differ from those of the control group,except for the second day of observation. The formulation according tothe present invention (DEAE/N-AC) instead produces better test scores,as of the third day, than baseline ones. The formulation thus seemscapable of effectively contrasting the effects of decreased lacrimalproduction caused by atropine.

[0090]FIG. 3 shows the results obtained through the observation, viaslit lamp, of the animals' cornea after staining with sodiumfluorescein. This colouring highlights alterations (comeal lesions)produced by AS treatment, of the kind normally found in eyes withdry-eye syndrome. For each treatment, the values are expressed as apercentage of eyes in which intensely coloured dots were noted(correposnding to epithelial alterations of the cornea) with respect tothe total number of eyes examined from the third to fifth day oftreatment. As may be noted, unlike with the commercially availableformulation, the formulation of Example 2 according to the presentinvention reduces the overall number of observed alterations practcallyto zero.

[0091] The present invention has been disclosed with particularreference to some specific embodiments thereof, but it should beunderstood that modifications and changes may be made by the personsskilled in the art without departing from the scope of the invention asdefined in the appended claims.

1. An ophthalmic composition based on acetylcysteine for administrationin eyedrops, containing N-acetyl-cysteine neutralized with baseDEAE-dextrane and having a physiologically acceptable osmolarity.
 2. Anophthalmic composition according to claim 1 wherein said osmolarity isless than 320 mOsm/kg.
 3. An ophthalmic composition according to claim 2having an osmolarity below 300 mOsm/kg and a pH between 6.0 and 7.5. 4.An ophthalmic composition according to claim 1 wherein said pH isbetween 6.2 and 7.0.
 5. An ophthalmic composition according to claim 1,containing, in water, from 3 to 5% by weight of N-acetyl-cysteine andfrom 9 to 15% by weight of DEAE-dextrane.
 6. An ophthalmic compositionaccording to claim 1, in the form of a solution or an aqueous suspensionin a pharmaceutically acceptable ophthalmic vehicle.
 7. An ophthalmiccomposition according to claim 6, also containing otherviscosity-enhancing polymers.
 8. An ophthalmic composition according toclaim 7 wherein said viscosity-enhancing polymers are selected from thegroup consisting of: polyvinyl alcohol, hydroxypropyl cellulose,hyaluronic acid, polyvinylpyrrolidone, chondroitin sulfate.
 9. Anophthalmic composition according to claim 6 also containingpreservatives, antimicrobials and/or chelating agents.
 10. An ophthalmiccomposition according to claim 1, containing 4% by weight ofN-acetyl-cysteine, 12% by weight of DEAE-dextrane, having a pH rangingbetween 6.2 and 7 and an osmolarity between 240 and 300 mOsm/kg.